The present invention relates to an expression element suitable for increasing the levels of expression of polypeptides in cells or organisms. In particular, the invention relates to an expression element having the sequence (SEQ ID NO: 7) CGGCAGGGTCTC.
Xylan, a heterogeneous polysaccharide commonly found in plant cell walls, is one of the most common polysaccharides in nature. Xylanase is one of the major enzymes involved in the breakdown of xylan, catalysing the digestion of xylan into oligoxylose subunits.
Xylanase enzymes and corresponding genes have been isolated from a very large number of different organisms. Examples include xylanases A and B from Penicillium, xylanase A from Thermotoga maritima and Bacillus subtilis, xylanases B and D from Ruminococcus flavefaciens, and many others.
It is generally established that it is desirable to direct expression of a heterologous nucleotide sequence in an organism, such as a filamentous fungus (e.g. Aspergillus niger), or yeast. The resultant protein or enzyme may then be used in industry. Alternatively, the resultant protein or enzyme may be useful for the organism itself. For example, it may be desirable to produce fungal protein products with an optimised amino acid composition and so increase the nutritive value thereof. For example, the fungus may be made more useful as a feed. In the alternative, it may be desirable to isolate the resultant protein or enzyme and then use the protein or enzyme to prepare, for example, food compositions. In this regard, the resultant protein or enzyme can be a component of the food composition or it can be used to prepare food compositions, including altering the characteristics or appearance of food compositions. It may even be desirable to use the organism, such as a filamentous fungus or a yeast, to express heterologous genes, such as for the same purposes.
Filamentous fungi in particular are attractive hosts for the large-scale production of proteins in industry. They have the capacity to secrete a large amount of heterologous and/or homologous protein into their growth medium, they have been extensively studied and are well known, and moreover they are considered safe for use in the preparation of products useful in the food, feed and beverage industries. They are known for their ability to secrete several hydrolytic enzymes, amongst which number the xylanases. A number of different xylanases are known and have been characterised and/or cloned (for example, see Ito, et al., (1992) Biosci. Biotech. Biochem. 56:906-912), including xylanases from A. awamori, A. kawachii and xylanases A, B and C from Aspergillus tubingensis. Of the latter, xylanase B is known to be more weakly expressed (see WO 94/14965). In general, xylanases belong to one of two families of glycosyl hydrolases, family H and family F. There is little homology between the two families, but within each family there is some sequence identity. For example, xylanases A and B of A. tubingensis belong to family H and share approximately 45% sequence identity at the amino acid level.
For heterologous expression in filamentous fungi, as well as the vast majority of other organisms, it is desirable to use a strong promoter to direct expression of the gene in question. Usually it is assumed that highly expressed genes contain a strong promoter and consequently promoters derived from highly expressed genes are frequently used for this purpose. Examples of expression systems for use in filamentous fungi include systems employing the glucoamylase promoter (U.S. Pat. No. 5,198,345) and the A. awamori xylanase A (xlnA) promoter (Gouka, et al., (1996) Appl. Microbiol. Biotechnol. 46, 28-35).
De Graaff et al., in xylans and xylanases, J. Visser et al. (eds.), 1992:235-246, Elsevier, and De Graaff et al., (1994) Molecular Microbiology 12:479-490, have identified a 158 bp upstream regulatory region of the A. tubingensis xlnA gene which is responsible for activation of transcription from the xlnA promoter. This region contains inter alia a sequence which comprises three repeats of the element (SEQ ID NO: 23) GTCCATTTAGCCA. De Graaff et al. showed that the entire 158 bp region was capable of activating an A. niger glucose oxidase gene (goxC) core promoter. It was not, however, determined whether the element (SEQ ID NO: 23) GTCCATTTAGCCA was itself responsible for the activity of the upstream region or the activating effect.
According to a first aspect of the present invention there is provided the use of a nucleic acid element having the sequence (SEQ ID NO: 7) CGGCAGGGTCTC to modulate transcription of a nucleotide sequence from a promoter.
Preferably, the promoter is a core promoter.
The present invention is concerned with the use of the sequence (SEQ ID NO: 7) CGGCAGGGTCTC as a control element for modulating transcription of a nucleotide sequence or nucleotide sequences from a promoter. The invention accordingly provides nucleic acid constructs in which at least one heterologous copy of the element is operatively linked to a promoter which is itself operatively linked to a nucleotide sequence, vectors containing such nucleic acid constructs and host cells transformed with such vectors andlor expressing DNA constructs according to the invention. Moreover, the invention concerns the use of multiple copies of the activating element operatively linked to the promoter, in order to provide further activation of transcription. In a further aspect, the invention concerns a sequence variant of the A. tubingensis xlnB gene which possesses enhanced expression characteristics as a result of the presence of three, rather than two, copies of the element of the invention upstream of the TATA box, as well as the use of this variant in nucleic acid constructs as above.
The element according to the invention is preferably placed upstream of a promoter which is operatively linked to a nucleotide sequence. The nucleotide sequence is preferably a heterologous nucleotide sequence.
Where a promoter already contains, in its upstream sequences, one or two copies of the regulatory element of the invention, an exogenous copy or copies is added in accordance with the invention in order to further activate transcription from this promoter.
The optimum number of elements is three. The presence of more than three elements is detrimental to transcriptional activation. The use of four or more elements is applicable where a lower, but still significant, level of transcription is desired. Thus, the invention may be applied to the modulation (downregulation as well as the upregulation) of transcription from a promoter. Preferably, the element of the invention is used to upregulate transcription from the promoter, such that the rate of transcription is increased.
Where more than one copy of the element of the invention is present, the elements may overlap. Thus, for example, the initial nucleotide of one element may also be the terminal nucleotide of another element. Preferably, the elements overlap by 1, 2 or 3 nucleotides. Preferably, the sequence of an overlapping element is (SEQ ID NO: 16) GGCAGGGTCTCGGCAGGGTCTC.
Preferably, the activated promoter linked to the nucleotide sequence according to the invention is incorporated into a nucleic acid construct, which may be a plasmid vector or the like. Advantageously, a vector according to the invention is an expression vector.
As stated above, three copies of the element provide the strongest transcriptional activation, being highly preferred to two elements. The addition of further elements, above three, leads to a progressive reduction in the activation level, as described in further detail in the accompanying examples.
Expression vectors according to the invention are useful for transforming cells. The invention accordingly provides cells transformed with vectors according to the invention, for the expression of polypeptides encoded by the heterologous nucleotide sequence.
The transformed cells may be cultured cells, for example in tissue culture or organ culture, but also cells which form all or part of a discrete living organism. The invention accordingly provides a transgenic non-human organism which has been transformed with a nucleic acid construct according to the invention. Such organisms may be unicellular or multicellular. Especially preferred are yeasts and filamentous fungi, especially Aspergillus.
In a second aspect of the present invention, there is provided a sequence variant of the A. tubingensis xlnB promoter, characterised in that it possesses at least three copies of the element (SEQ ID NO: 7) CGGCAGGGTCTC. The xylanase B gene is weakly expressed (WO 94/14965) and on account of its being seen as possessing a weak promoter has never been suggested to be a suitable basis for heterologous expression systems. A surprisingly effective sequence variant of the xylanase B promoter, which is particularly strong and suitable for the expression of heterologous nucleotide sequences, has now been isolated and compared to the promoter disclosed in WO 94/14965. The new sequence variant differs from the promoter of WO 94/14965 in the presence of three copies of the element (SEQ ID NO: 7) CGGCAGGGTCTC rather than just two. The presence of the extra element is through to be responsible for the observed increase in transcriptional activation.
The preferred sequence of the variant according to the invention is that of the xlnB promoter as hereinbefore defined shown in SEQ. ID. No. 1, or a variant, homologue or fragnent thereof, provided that the variant, homologue or fragment retains at least three copies of the element according to the invention. The promoter in SEQ. ID. No. 1 is located upstream of the translation start site, and in a preferred embodiment the promoter possesses the sequence as shown between positions 1 and 720 of SEQ. ID. No. 1. Advantageously, the promoter possesses a shorter sequence, preferably that shown between positions 342 and 720 of SEQ. ID. No. 1.
The sequence variant according to the invention may be incorporated in a nucleic acid molecule such that it is operatively linked to a nucleotide sequence. The sequence variant has advantageous properties as a promoter for gene expression, being stronger that the xlnB promoter published in WO 94/14965, and is useful for the expression of a variety of polypeptides, including those encoded by homologous nucleotide sequences such as xylanase B.
The invention is particularly useful in the production of substances useful in the foodstuffs, feed and beverage industries. The preferred host, Aspergillus, is a popular production host in these industries, for the reasons set forth hereinbefore.
The invention moreover provides a method for increasing the level of transcription of a nucleotide sequence from a promoter comprising the steps of inserting at least one heterologous copy of the element (SEQ ID NO: 7) CGGCAGGGTCTC such that it is operably linked to the promoter, and causing the nucleotide sequence to be transcribed.
As used herein, the term xe2x80x9cnucleic acidxe2x80x9d includes to the natural nucleic acids DNA and RNA, or synthetic nucleic acid analogues which share at least one of the properties of natural nucleic acids, such as the ability to encode a protein or the ability to hybridise to other nucleic acid molecules, preferably natural nucleic acid molecules. The preferred nucleic acid for use in the invention is DNA. Coding sequences may preferably comprise cDNA.
References to the element (SEQ ID NO: 7) CGGCAGGGTCTC according to the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleotides from or to the sequence providing the resultant nucleotide sequence has the ability to act as an activating element when operatively linked to a promoter in an expression system. In particular, the invention covers homologues of the sequence (SEQ ID NO: 7) CGGCAGGGTCTC as evidenced by relative identity in sequence and/or structure and/or function providing the resultant nucleotide sequence has the ability to act as an activating element according to the invention. With respect to sequence xe2x80x9chomologyxe2x80x9d, preferably there is sequence identity of at least 75%, more preferably at least 85%, more preferably at least 90% to the sequence (SEQ ID NO: 7) CGGCAGGGTCTC. More preferably the element of the invention has no more than 1, 2 or 3 nucleotide alterations from the sequence (SEQ ID NO: 7) CGGCAGGGTCTC. Most preferably the element has the sequence (SEQ ID NO: 7) CGGCAGGGTCTC.
The sequences which form part of the present invention may also be in the form of complementary sequences. The term xe2x80x9ccomplementaryxe2x80x9d means that the present invention also covers nucleotide sequences that can hybridise to the nucleotide sequences of the nucleotide sequence or the promoter sequence, respectively.
The element according to the invention is capable of potentiating transcription from a promoter. This means that, in the presence of the element, the potential for transcription is increased. The promoter may remain subject to other influences, such as tissue specific or nutrient-influenced transcriptional regulation, which may so control the transcription from the promoter that no effect is seen with the addition of the element of the invention. However, when such further influences are neutralised, the element will cause an increase in the level of transcription from the promoter.
The term xe2x80x9cconstructxe2x80x9d is synonymous with terms such as xe2x80x9cconjugatexe2x80x9d, xe2x80x9ccassettexe2x80x9d and xe2x80x9chybridxe2x80x9d. Constructs may be prepared by linking nucleic acid sequences directly or indirectly, according to techniques known in the art. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Sh1-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term xe2x80x9cfusedxe2x80x9d in relation to the present invention which includes direct or indirect attachment.
The term xe2x80x9cvectorxe2x80x9d includes expression vectors and transformation vectors.
The term xe2x80x9cexpression vectorxe2x80x9d means a construct capable of in vivo or in vitro expression of a polypeptide gene product encoded by a coding sequence inserted into the vector.
The term xe2x80x9ctransformation vectorxe2x80x9d means a construct capable of being transferred from one species to anotherxe2x80x94such as from an E. coli plasmid to a filamentous fungus, preferably of the genus Aspergillus.
As used herein, xe2x80x9cvectorxe2x80x9d refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are known to those skilled in the art. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA amplification or for expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.
Both expression and cloning vectors generally contain at least one nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Typically in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and other fungi. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2xcexc plasmid origin is suitable for yeast, and fungal origins may be employed in filamentous fungi, for example the amal replicon (Gems et al., (1991) Gene 98:61-67).
Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or other fungal cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be replicated by insertion into the host genome.
Advantageously, an expression and cloning vector may contain a selection gene also referred to as selectable marker which allows for the selection of the genetic construct in, for example, a filamentous fungus, preferably of the genus Aspergillus, such as Aspergillus niger, into which it has been transferred. This gene may encode a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from the medium.
As to a selective gene marker appropriate for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene. Suitable markers for yeast are, for example, those conferring resistance to antibiotics G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2, TRP1, or HIS3 gene.
Since the replication of vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript(copyright) vector or a pUC plasmid, e.g. pUC18 or pUC19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.
Moreover, the vector according to the invention preferably includes a secretion sequence in order to facilitate secretion of the polypeptide from hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body.
Construction of vectors according to the invention employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing expression and function are known to those skilled in the art. Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.
The term xe2x80x9chost cellxe2x80x9d, as used herein, includes any cell type, whether a unicellular organism, a cell derived from a multicellular organism and placed in tissue culture or a cell present as part of a multicellular organism, which is susceptible to transformation with a nucleic acid construct according to the invention. Such host cells, such as yeast and higher eukaryote cells, fungal cells and plant cells may be used for replicating DNA and producing polypeptides encoded by nucleotide sequences as used in the invention. Prokaryotic cells are suitable for replicating DNA and include eubacteria, such as Gram-negative or Gram-positive organisms, such as E. coli, e.g. E. coli K-12 strains, DH5xcex1 and HB101, or Bacilli. Host cells suitable for replicating nucleic acids and expressing coding sequences encoded on vectors according to the invention include eukaryotic microbes such as filamentous fungi, e.g. Aspergillus, or yeast, e.g. Saccharomyces.
Particularly preferred are cells from filamentous fungi, preferably Aspergillus, such as A. niger, A. awamori and A. tubingensis. 
DNA may be stably incorporated into cells or may be transiently expressed using methods known in the art. Stably transfected cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient transfectants, cells are transfected with a reporter gene to monitor transfection efficiency.
Host cells are transfected or, preferably, transformed with expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing expression, selecting transformants, or amplifying the genes encoding the desired sequences. Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique or by electroporation. Numerous methods of transfection are known to the skilled worker in the field. Successful transfection is generally recognised when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used.
Incorporation of cloned DNA into a suitable expression vector, transfection of eukaryotic cells with a plasmid vector or a combination of plasmid vectors, each encoding one or more distinct genes or with linear DNA, and selection of transfected cells are well known in the art (see, e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press) and set forth in particular on pages 15 and 16 herein.
Transfected or transformed cells are cultured using media and culturing methods known in the art, preferably under conditions, whereby the polypeptide encoded by the heterologous nucleotide sequence is expressed. The composition of suitable media is known to those in the art, so that they can be readily prepared. Suitable culturing media are also commercially available.
The term xe2x80x9corganismxe2x80x9d in relation to the present invention includes any organism that could express a nucleotide sequence under the control of the promoter according to the present invention. Organisms in which the molecules according to the invention are expressed are preferably fungal hosts.
Preferably the organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably A. niger, A. awamori or A. tubingensis. 
Other preferred organisms include any one of Aspergillus oryzae, Trichoderma reesei, T. viride and T. longibrachiatum. 
The term xe2x80x9ctransgenic organismxe2x80x9d in relation to the present invention includes any organism that comprises the promoter according to the present invention and a nucleotide sequence coding for a heterologous nucleotide sequence, wherein the nucleotide sequence according to the present invention can be expressed within the organism. Preferably the promoter and the nucleotide sequence are incorporated in the genome of the organism.
The term xe2x80x9ctransgenic organismxe2x80x9d does not cover the native nucleotide sequence according to the present invention in its natural environment when it is under the control of its native promoter which is also in its natural environment. In addition, the present invention does not cover the native enzyme according to the present invention when it is in its natural environment and when it has been expressed by its native nucleotide sequence which is also in its natural environment and when that nucleotide sequence is under the control of its native promoter which is also in its natural environment.
The transformed cell or organism may be used to prepare acceptable quantities of the desired compound which may be easily retrievable from the cell or organism.
The term xe2x80x9cpromoterxe2x80x9d is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression. Preferably, fungal promoters are used in the present invention. Any suitable promoter may be used in connection with the present invention, with xylan-inducible promoters preferably of fungal origin being especially indicated. A xe2x80x9ccorexe2x80x9d promoter, as referred to herein, in a promoter consisting essentially of a TATA box and a transcriptional initiation site.
Fungal promoters are known in the literature (for example, see Gurr, et al., (1987) The structure and organisation of nuclear genes of filamentous fungi. In Kinghorn, J. R. (ed), Gene Structure in Eukaryotic Microbes, IRL Press, Oxford, pp. 93-139).
In addition to the nucleotide sequences described above, the promoter of the present invention can additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as a TATA box. The TATA box is typically found 30 bp upstream of the transcription initiation site, and is believed to be involved in the assembly of the transcriptional complex.
The promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the heterologous nucleotide sequence. Fungal promoters, for instance, typically contain both a TATA box and a UAS (Upstream Activating Site). The UAS is a binding site for the activating regulator acting on the promoter in question. The element of the present invention is believed to be a UAS. Other sites, involved in various aspects of promoter regulation, may also be included.
The element of the invention is, according to the present invention, placed upstream of the promoter. The distance between the promoter and the element may be adjusted, by empirical experimentation, to maximise or adjust the level of transcriptional activation.
The term xe2x80x9cheterologous nucleotide sequencexe2x80x9d with reference to the constructs according to the present invention means any sequence encoding a polypeptide of interest, other than the complete natural sequence normally associated with the promoter employed, or a sequence which is capable of expressing a nucleic acid, for example a regulatory RNA such as an antisense RNA or a ribozyme, or a tRNA or rRNA capable of regulating the metabolism of an organism. Conversely, the term xe2x80x9cnucleotide sequencexe2x80x9d includes homologous nucleotide sequences which are the sequences normally associated with the promoters employed. The invention includes the use of both homologous and heterologous nucleotide sequences.
A heterologous nucleotide sequence can be any nucleotide sequence that is either foreign or natural to the organism (e.g. filamentous fungus, preferably of the genus Aspergillus, or a plant) in question. The term xe2x80x9cheterologous nucleotide sequencexe2x80x9d also includes a homologous nucleotide sequence which has been mutated, such as by insertion, addition, deletion or alteration, such that it is no longer identical with the natural homologous nucleotide sequence.
Typical examples of a heterologous nucleotide sequence include sequences coding for proteins and enzymes that modify metabolic and catabolic processes. The heterologous nucleotide sequence may code for an agent for introducing or increasing pathogen resistance. The heterologous nucleotide sequence may be an antisense construct for modifying the expression of natural transcripts present in the relevant tissues. The heterologous nucleotide sequence may code for a non-native protein of a filamentous fungus, preferably of the genus Aspergillus, or a compound that is of benefit to animals or humans. Examples of nucleotide sequences encoding enzymes include pectinases, pectin depolymerases, polygalacturonases, pectate lyases, pectin lyases, hexose oxidase, oxidoreductases, lipases, glucan lyase, rhamno-galacturonases, hemicellulases, endo-xcex2-glucanases, arabinases, or acetyl esterases, or combinations thereof, as well as antisense sequences thereof. The heterologous nucleotide sequence may be a protein giving nutritional value to a food or crop. Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition (e.g. a higher lysine content than a non-transgenic plant).
The heterologous nucleotide sequence may code for an enzyme that can be used in food processing such as chymosin, thaumatin, xcex1-galactosidase, a glucanase or xcex2-1,4-endoglucanase.
The heterologous nucleotide sequence may code for an intron of a particular nucleotide sequence, wherein the intron can be in sense or antisense orientation.
The heterologous nucleotide sequence can be the nucleotide sequence coding for the arabinofuranosidase enzyme which is the subject of PCT patent application PCT/EP96/01009 (incorporated herein by reference). The heterologous nucleotide sequence can be any of the nucleotide sequences coding for the ADP-glucose pyrophosphorylase enzymes which are the subject of PCT patent application PCT/EP94/01082 (incorporated herein by reference). The heterologous nucleotide sequence can be any of the nucleotide sequences coding for the xcex1-glucan lyase enzyme which are described in PCT patent application PCT/EP94/03397 (incorporated herein by reference). The heterologous nucleotide sequence can be any of the sequences coding for T. lanuginosus amylase, as described in PCT patent application PCT/EP95/02607, incorporated herein by reference. The heterologous nucleotide sequence can be any of the nucleotide sequences coding for the glucanase enzyme which are described in PCT patent application PCT/EP96/01008 (incorporated herein by reference). The heterologous nucleotide sequence can be xylanase A or B, as set forth herein.
Preferably the promoter and the nucleotide sequence according to the invention are stably maintained within host cells or transgenic organisms. By way of example, the promoter and/or the nucleotide sequence may be maintained within the transgenic organism in a stable extrachromosomal construct. This is preferred for transgenic yeast or some filamentous fungi. Alternatively, the promoter and/or the heterologous nucleotide sequence (such as the nucleotide sequence according to the present invention) may be stably incorporated within the transgenic organism""s genome. This is preferred for some transgenic yeast, and most filamentous fungi.
A preferred host organism for the expression of the nucleic acid constructs of the present invention and/or for the preparation of the heterologous polypeptides according to the present invention is an organism of the genus Aspergillus, such as Aspergillus niger. In this regard, a transgenic Aspergillus according to the present invention can be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-393), Davis R. W. 1994 (Heterologous gene expression and protein secretion in Aspergillus. In: Martinelli S. D., Kinghorn J. R. (Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp 525-560), Ballance, D. J. 1991 (Transformation systems for Filamentous Fungi and an Overview of Fungal Gene structure. In: Leong, S. A., Berka R. M. (Editors) Molecular Industrial Mycology. Systems and Applications for Filamentous Fungi. Marcel Dekker Inc. New York 1991. pp 1-29) and Turner G. 1994 (Vectors for genetic manipulation. In: Martinelli S. D., Kinghorn J. R.(Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641-666). The following commentary provides a summary of those teachings for producing transgenic Aspergillus according to the present invention.
For almost a century, filamentous fungi have been widely used in many types of industry for the production of organic compounds and enzymes. For example, traditional Japanese koji and soy fermentations have used Aspergillus sp. Also, in this century Aspergillus niger has been used for production of organic acids particular citric acid and for production of various enzymes for use in industry.
There are two major reasons why filamentous fungi have been so widely used in industry. First filamentous fungi can produce high amounts of extracelluar products, for example enzymes and organic compounds such as antibiotics or organic acids. Second filamentous fungi can grow on low cost substrates such as grains, bran, beet pulp etc. The same reasons have made filamentous fungi attractive organisms as hosts for heterologous expression according to the present invention.
In order to prepare the transgenic Aspergillus, expression constructs are prepared by inserting a heterologous nucleotide sequence (such as a nucleotide sequence coding for an amylase enzyme) into a construct designed for expression in filamentous fungi.
Several types of constructs used for heterologous expression have been developed. The constructs contain the promoter according to the present invention which is active in fungi. The heterologous nucleotide sequence can be fused to a signal sequence which directs the protein encoded by the heterologous nucleotide sequence to be secreted. Usually a signal sequence of fungal origin is used. A terminator active in fungi may also be employed.
Another type of expression system has been developed in fungi where the heterologous nucleotide sequence is fused to a fungal gene encoding a stable protein. This can stabilise the protein encoded by the heterologous nucleotide sequence which encodes a protein of interest (POI). In such a system a cleavage site, recognised by a specific protease, can be introduced between the fungal protein and the protein encoded by the heterologous nucleotide sequence, so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the protein encoded by the heterologous nucleotide sequence. By way of example, one can introduce a site which is recognised by a KEX-2 like peptidase found in at least some Aspergilli (Broekhuijsen et al 1993 J Biotechnol 31 135-145). Such a fusion leads to cleavage in vivo resulting in protection of the expressed product and not a larger fusion protein (see U.S. Pat. No. 5,679,543).
Heterologous expression in Aspergillus has been reported for several genes coding for bacterial, fungal, vertebrate and plant proteins. The proteins can be deposited intracellularly if the nucleotide sequence according to the present invention (or another heterologous nucleotide sequence) is not fused to a signal sequence. Such proteins will accumulate in the cytoplasm and will usually not be glycosylated which can be an advantage for some bacterial proteins. If the nucleotide sequence according to the present invention (or another heterologous nucleotide sequence) is equipped with a signal sequence the protein will accumulate extracelluarly.
With regard to product stability and host strain modifications, some heterologous proteins are not very stable when they are secreted into the culture fluid of fungi. Most fungi produce several extracelluar proteases which degrade heterologous proteins. To avoid this problem special fungal strains with reduced protease production have been used as host for heterologous production.
For the transformation of filamentous fungi, several transformation protocols have been developed for many filamentous fungi (Ballance, D. J. 1991 (Transformation systems for Filamentous Fungi and an Overview of Fungal Gene structure. In: Leong, S. A., Berka R. M. (Editors) Molecular Industrial Mycology. Systems and Applications for Filamentous Fungi. Marcel Dekker Inc. New York 1991. pp 1-29). Many of them are based on preparation of protoplasts and introduction of DNA into the protoplasts using PEG and Ca2+ ions. The transformed protoplasts then regenerate and the transformed fungi are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as argB, trpC, niaD and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance. A commonly used transformation marker is the amdS gene of A. nidulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source.
In another embodiment the transgenic organism can be a yeast. In this regard, yeast have also been widely used as a vehicle for heterologous gene expression. The species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarranton, eds, pp 107-133, Blackie, Glasgow).
For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae. 
A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993. xe2x80x9cYeast as a vehicle for the expression of heterologous genesxe2x80x9d, Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting a nucleotide sequence comprising a DNA construct according to the present invention into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed, suitable component parts of which are discussed hereinbefore.
For the transformation of yeast several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
The following sequences are set forth in the Sequence Listing:
Deposition Data
The following deposits have been made under the Budapest Treaty in connection with the present application at the NCIMB, 23 St. Machar Drive, Aberdeen, AB2 1RY, Scotland:
The invention is described hereinbelow, for the purposes of illustration only, with reference to the following examples, in which reference is made to the following figures.
FIG. 1 shows plasmid pxlnB-AmyA, which comprises the xlnB promoter, the amyA gene and the xlnA terminator, together with an ampicillin resistance selection marker.
FIG. 2 shows a mono-Q chromatogram of the culture broth of A. niger transformed with pxlnB-AmyA.
FIG. 3 shows plasmid LMJterm.
FIG. 4 shows plasmid pPR66.
FIG. 5 shows plasmid pPR67.
FIG. 6 shows plasmid pPR68.
FIG. 7 shows plasmid pPR69.
FIG. 8 shows plasmid pPR70.
FIG. 9 shows a silver-stained gel of the products derived from A. niger transformed with pPR70 and pPR70-6.
FIG. 10 shows a plot of the results of the cultivation of A. tubingensis transformed with a xlnB-xlnA expression construct.
FIG. 11 shows the alignment of Ppr70 and variants among the xlnB elements. The start of the sequences is the SnaBI site (5661) into which the elements are inserted.